Cancer cells capable of tumorigenesis but incapable of infiltration and metastasis, process for preparing the same, and method for screening gene relating to metastasis by using the same

ABSTRACT

Tumor cells having tumorigenic potential but lacking invasive/metastatic potential are established by introducing an oncogene of the ras family into a BALB/c 3T3 A31-variant cell. A screening method for genes having the property of conferring invasive/metastatic potential is also provided, which comprises transfecting DNA derived from a tumor tissue obtained from the surface or inside of a mammal or derived from a tumor cell line into said tumor cells having tumorigenic potential but lacking invasive/metastatic potential, isolating cells having acquired invasive/metastatic potential and extracting DNA therefrom.

FIELD OF THE INVENTION

[0001] The present invention relates to tumor cells having tumorigenicpotential but lacking invasive/metastatic potential, a method forpreparing them and a screening method for metastasis-related genes usingthem.

PRIOR ART

[0002] Invasion and metastasis occur at the final stage of tumorprogression and are the major cause of death of cancer patients.However, it is difficult to treat such effects because the molecularmechanism by which malignant phenotypes of tumor cells such asinvasion/metastasis are expressed has been mostly unknown. Although theexpression of malignant phenotypes such as invasion/metastasis has longbeen recognized as a biological phenomenon which is pathologicallydistinguished from tumor formation in carcinogenic process, there hasbeen only limited information about biochemical basis therefor andtarget groups of molecules for therapy have not been well identified.

[0003] Generally, accumulation of alterations of genetic information ofoncogenes and tumor suppressor genes is thought to make cells cancerousand sometimes invasive/metastatic. Namely, a gene that plays a role inmediating an external signal into the nucleus is mutated to form a geneproduct, which activates the downstream signaling cascade continuously.This causes phenotypes of tumor cells to be expressed. It is alsothought that tumor cells become invasive/metastatic during tumorprogression as a result of additional genetic alterations to thoserequired for tumorigenicity (Evans C. W. The Metastatic Cell: Behaviorand Biochemistry, Chapman & Hall, London, 1991). Therefore, metastasisof tumor cells may be induced by an aberrant intracellular signalingsystem that is different from the signaling system required fortumor-igenicity. Thus, it is very important for investigations of themechanism of invasion/metastasis to elucidate a metastasis-specifictumor-related signaling pathway that is responsible for tumor cellsbecoming invasive/metastatic.

[0004] For such investigations, a cell which could separably acquiretumorigenic potential and invasive/metastatic potential by oncogenetransfer would be useful for identifying the above pathway and moleculesinvolved therein.

[0005] A set of two cells derived from the same parent strain, one ofwhich has tumorigenic potential but lacks invasive/metastatic potentialwhile the other has both potentials, would be useful for investigatingthe signaling pathway involved in metastasis. Some tumor cell linessatisfying this criterion have already been established. They includerodent-derived cell lines K1735, B16, T-lymphoma L5178Y, Lewis lungcarcinoma and rat ascites hepatoma AH7974, as well as their derivativesobtained by in vitro culture and selection of cells recovered from invivo metastases of these cell lines (Fidler I. J. et al., J. Natl.Cancer Inst. 67:947-956, 1981; Talmadge J. E. et al., J. Natl. CancerInst. 69:975-980, 1982; Fidler I. J., Nature 242:148-149, 1973; FidlerI. J., Cancer Res. 35:218-224, 1975; Fidler I. J. et al., Am. J. Pathol.97:633-648, 1979; Ota et al., Clin. Exp. Metastasis 10:297-308, 1992;Olsson L. et al., Cancer Res. 41:4706-4709, 1981; Brodt P., Cancer Res.46:2442-2448, 1986; Pal. K. et al., Invasion Metastasis 5:159-169, 1985;Young M. R. et al., Cancer Res. 45:3918-3923, 1985; Kawaguchi T. et al.,Clin. Exp. Metastasis 10:225-238, 1992). However, phenotypes of thesecell lines are sometimes unstable during experimentations (Poste G. etal., Proc. Natl. Acad. Sci. USA 78:6226-6230, 1981) and many propertiesof these cell lines that are not related to the acquisition ofinvasive/metastatic potential may change during in vivo selection.

[0006] It would be useful and valuable if a stable set of cell lines asdescribed above could be obtained by oncogene transfer. However, whetheror not cells acquire tumorigenic potential and invasive/metastaticpotential by oncogene transfer depends on the nature of the cell lineused (Muschel R. J. et al., Am. J. Pathol. 121:1-8, 1985). Moreover,oncogene transfer frequently induces invasive/metastatic potential inrecipient cells to a greater or lesser extent simultaneously withtumorigenicity (Thorgeirsson U. P., Mol. Cell. Biol. 5:259-262, 1985;Egan S. E. et al., Science 238:202-205, 1987). Up to the present, noreport has shown exact establishment of a cell line having tumorigenicpotential but lacking invasive/metastatic potential and a cell linehaving both of these potentials. Thus, it was not easy to separately usecharacteristics of tumorigenic potential and invasive/metastaticpotential of tumor cells for investigations in an experimental system.

[0007] It is an object of the present invention to establish a tumorcell line having tumorigenic potential but lacking invasive/metastaticpotential, which is suitable for investigating the difference betweenthe signaling pathway for acquiring tumorigenic potential and thesignaling pathway for acquiring invasive/metastatic potential. Such atumor cell line can be used in combination with a cell line derived fromthe same parent strain but having both of tumorigenic andinvasive/metastatic potentials for investigations of characteristics ofinvasive/metastatic potential. It is another object of the presentinvention to develop a screening method for tumor metastasis-relatedgenes using the tumor cell line of the present invention.

SUMMARY OF THE INVENTION

[0008] As a result of careful studies to overcome the above problems, wesucceeded in establishing a tumor cell line having tumorigenic potentialbut lacking invasive/metastatic potential by introducing a specificoncogene into a recipient cell.

[0009] Accordingly, the present invention provides a tumor cell havingtumorigenic potential but lacking invasive/metastatic potential. Apreferred tumor cell of the present invention having such properties is1-1ras1000 deposited with the National Institute of Bioscience andHuman-Technology of the Agency of Industrial Science and Technologyunder accession number FERM BP-5406.

[0010] The present invention also provides a method for preparing saidtumor cell having tumorigenic potential but lacking invasive/metastaticpotential by introducing an oncogene of the ras family into a BALB/c 3T3A31 variant cell.

[0011] The present invention also provides a screening method for geneshaving the property of conferring invasive/metastatic potential, whichcomprises transfecting DNA derived from a tumor tissue obtained from thesurface or inside of a mammal or derived from a tumor cell line into atumor cell having tumorigenic potential but lacking invasive/metastaticpotential, isolating cells having acquired invasive/metastatic potentialand extracting DNA therefrom.

BRIEF DESCRIPTION OF THE DRAWINGS

[0012]FIG. 1. The copy number of introduced mutated c-Ha-ras gene byslot blot hybridization in ras-transformed A31 variant cells(electrophoretic photographs).

[0013]FIG. 2. Immunoblotting analysis of ras-transformed A31 variantcells using an anti-p21^(ras) monoclonal antibody NCC-RAS-004(electrophoretic photographs). Lane 1: 5 ng of standard proteinp21Gly-12 corresponding to the product of normal human c-Ha-ras1 plus 5μl of lysate from T24 bladder carcinoma cells carrying mutated c-Ha-raslgene; Lane 2: 5 μl of lysate from A31-1-1 cells: Lane 3: 50 μl (10-foldexcess) of lysate from A31-1-13 cells; Lane 4: 5 μl of lysate fromA31-1-1ras cells; Lane 5: 5 μl of lysate from 1-1ras1000 cells; Lane 6:5 μl of lysate from A31-1-13 cells; Lane 7: 5 μl of lysate from1-13ras1000 cells.

[0014]FIG. 3. Immunoblotting analysis (A) and phosphorylation assay (B)of v-src-transformed BALB/c 3T3 A31 variant cells (electrophoreticphotographs). Lane 1: 1-1; Lane 2: 1-13; Lanes 3-6: 1-1-derivedsrc-transformants; Lanes 7-10: 1-13-derived src-transformants.

[0015]FIG. 4. Invasive or metastatic potentials of ras-transformed1-1ras1000 and src-transformed 1-1src cells in either wild-type orNK-suppressed BALB/c mice (morphologic photographs the organisms).

[0016]FIG. 5. In vitro invasion assayed by counting the number of cellspenetrating Matrigel-coated filters using a microscope of 400magnifications (A), and cell motility as the motility index measured bycomputer-aided digital image analysis (B). Each column indicates themean of triplicate samples with standard deviation. Column 1: 1-ras;Column 2: 1-1ras1000; Column 3: 1-13ras; Column 4: 1-13ras1000, Column5: 1-1src; Column 6: 1-13src.

DETAILED DESCRIPTION OF THE INVENTION

[0017] Suitable recipient cells for preparing a cell line havingtumorigenic potential but lacking invasive/metastatic potential maypreferably be selected so that the resulting cell line can be used toprepare a counterpart of the above set of cell lines, i.e. a cell linehaving both of tumorigenic potential and invasive/metastatic potential.In this respect, suitable recipient cells for oncogene transfer shouldhave the following properties:

[0018] 1) they should have a stable non-metastatic phenotype;

[0019] 2) they should be able to afford a cell which has acquiredinvasive/metastatic potential by oncogene transfer;

[0020] 3) they should be able to select a cell which has acquiredinvasive/metastatic potential by oncogene transfer.

[0021] Recipient cells satisfying these properties include but are notlimited to, for example, BALB/c 3T3 A31 cells (Kakunaga T., et al.(eds.) Transformation Assay of Established Cell Lines: Mechanisms andApplication, Oxford University Press, New York, 1985, pp. 55-73), andother cells having the above properties. BALB/c 3T3 A31 cells include,for example, BALB/c 3T3 A31-1-1, BALB/c 3T3 A31-1-8 and BALB/c 3T3A31-1-13 established by Kakunaga as used in the examples of the presentinvention (Kakunaga et al., Science 209:505-507, 1980).

[0022] The oncogene to be introduced into a recipient cell may be anoncogene which allows the recipient cell to form a focus (focusformation or a swell of cells means cancerization of cells) and has theactivity of conferring no invasive/metastatic potential, such as a geneof the ras family. According to the present invention, tumor cellshaving tumorigenic potential but lacking invasive/metastatic potentialcould be established particularly by using, but not limited to, theactivated c-Ha-ras.

[0023] Tumor cells of the present invention are prepared by providing aplasmid carrying said oncogene and introducing it into a recipient cell.A convenient method for introducing an oncogene into a recipient cell iselectroporation (Tatsuka et al., Exp. Cell Res. 178:154-162, 1988), butother means such as the calcium phosphate method, microinjection mayalso be used.

[0024] Transformed cells are seeded in dishes to assay focus formation,and arising transformed foci are isolated and multiplied as anindependent cell strain or pooled.

[0025] Correct integration of the oncogene in the transformed cells canbe ascertained by Southern blot analysis, while transcription of theintegrated gene into mRNA can be ascertained by Northern blot analysisof the transcribed mRNA. Furthermore, translation of mRNA into proteincan be ascertained by immunoblotting analysis using, for example, ananti-p21^(ras) antibody.

[0026] An animal is used to evaluate tumorigenic potential andinvasive/metastatic potential of thus obtained cell line. In the presentinvention, BALB/c nude mice were used. In tumorigenicity assay,transformed cells were subcutaneously injected into the nude mice toobserve whether or not a tumor is formed, and growth rate and latencyperiod (the period from injection to detection of a tumorous nodule). Inexperimental metastasis assay, transformed cells were injected into thetail vein of the nude mice, and after 5 weeks, the mice were sacrificedand autopsied for metastasis.

[0027] The present invention thus succeeded in establishing cell lineshaving tumorigenic potential but lacking invasive/metastatic potentialas described above. A particularly preferable cell line established bythe present invention was designated as 1-1ras1000 and internationallydeposited with the National Institute of Bioscience and Human-Technologyof the Agency of Industrial Science and Technology (residing at 1-3,Higashi 1-Chome, Tsukuba-city, Ibaraki-prefecture, 305 Japan) on Feb.20, 1996 under accession number FERM BP-5406. 1-13ras1000 is anotherpreferable tumor cell line having similar properties to those of1-1ras1000.

[0028] 1-1ras1000 is a clone obtained by amplification to enhance theexpression level of the oncogene introduced, in which the ras gene isoverexpressed at about 16-fold amplification of the parent strainisolated from foci. The non-metastatic phenotype of 1-1ras1000 cells isso stable that they can not be turned metastatic even after in vitropassage or treatment with initiators such as UV rays ormethylcholanthrene or treatment with TPA. It is very difficult to obtainan invasive/metastatic clone from these cells by selection involvingpermeation through micropore filters coated with basement membrane(Matrigel).

[0029] Thus, 1-1ras1000 cells have a very stable non-metastaticphenotype without expressing metastatic phenotype. However, it ispossible that this property may result from potential lack ofinvasive/metastatic potential in 1-1ras1000 cells. The src oncogeneintroduced into these cells induced a great number of metastases inlung. This proved that 1-1ras1000 cells do not lack the step ofacquiring invasive/metastatic potential. The cells that took upinvasive/metastatic src showed a higher motility than the cells thattook up ras on basement membrane (Matrigel). Also in experiments usingmicropore filters, src-transformed cells were invasive for basementmembrane.

[0030] The present invention thus provides a set of a tumor cell linehaving tumorigenic potential but lacking invasive/metastatic potentialand a tumor cell line having both of tumorigenic potential andinvasive/metastatic potential as described above, allowingcharacteristics of invasive/metastatic potential and tumorigenicpotential to be separately used for investigations.

[0031] Cell lines having tumorigenic potential but lackinginvasive/metastatic potential of the present invention can be used forscreening for genes conferring invasive/metastatic potential. Thescreening method for genes having the property of conferringinvasive/metastatic potential according to the present invention can beperformed by transfecting DNA derived from a tumor tissue obtained fromthe surface or inside of a mammal or derived from a tumor cell line intoa cell line of the present invention, isolating cells having acquiredinvasive/metastatic potential and extracting DNA therefrom.

[0032] A specific example of the screening method for genes having theproperty of conferring invasive/metastatic potential using strain1-1ras1000 of the present invention can be performed by the followingprocedures:

[0033] 1) Prepare a genomic gene of a metastatic tumor cell (derivedfrom a tumor tissue obtained from the surface or inside of a mammal orderived from a tumor cell line) or a cDNA library (derived from a tumortissue obtained from the surface or inside of a mammal or derived from atumor cell line) constructed with a mammalian cell expression vector.

[0034] 2) Introduce either the genomic gene or cDNA library obtainedin 1) and a selectable marker (for example, a hygromycin-resistant gene)into 1-1ras1000 cells, by using the calcium phosphate method,electroporation, lipofection or the like. Other selecting means such asneo-resistant genes or gpt selection may also be used.

[0035] 3) If the selectable marker is a hygromycin-resistant gene, growthe cells in a medium containing hygromycin to affordhygromycin-resistant cells.

[0036] 4) Select hygromycin-resistant cells on the basis ofinvasive/metastatic potential.

[0037] 5) Recover non-c-Ha-ras gene fragments containing humangene-specific Alu repeats by using, for example, plaque hybridization.

[0038] If a cDNA library prepared with a mammalian cell expressionvector was introduced, gene fragments can be recovered as episomes(Tatsuka M. et al., Nature 359:333-336, 1992) or using plasmid rescue,PCR or library construction.

[0039] 6) Introduce thus recovered gene fragments alone or in mixtureinto 1-1ras1000 cells and examine invasive/metastatic potential.Specifically, for example, phage DNA obtained by plaque hybridizationmay be multiplied for each clone and transfected alone or in mixtureinto 1-1ras1000 cells, and the transfected cells may be injectedsubcutaneously or into the tail vein of nude mice to obtain a phageclone harboring metastatic cells.

[0040] 7) Isolate nearly full-length CDNA from the CDNA library using agene conferring invasive/metastatic potential as a probe and determineits nucleotide sequence. Determine the amino acid sequence on thisbasis.

[0041] 8) Screen a human genomic gene library using the cDNA isolated in7) as a probe to isolate human genomic genes containing all the exons.

[0042] 9) Integrate the cDNA isolated in 7) into a mammalian cellexpression vector and transfect it into 1-1ras1000 cells to examineinvasive/metastatic potential.

[0043] Thus obtained genes can be regarded as genes conferringinvasive/metastatic potential on tumor cells.

[0044] Various studies have previously been devoted to screening formetastasis-related genes. For example, Weiberg's group tried expressioncloning of a metastatic gene using NIH3T3 cells with failure (BernsteinS. C. and Weiberg R., Proc. Natl. Acad. Sci. USA 82:1726-1730, 1985).Genes such as nm23 (Leone A. et al., Cell 65:25-35, 1991), CD44(Gunthert U. et al., Cell 65:13-24, 1991), Kai-1 (Dong J. T. et al.,Science 268:884-886, 1995) have been cloned by a process involvingextracting mRNA from highly metastatic tumor cells while separatelyextracting mRNA from less metastatic tumor cells and comparing bothmRNAs to identify mRNA prevailing in highly metastatic tumor cells.Alternatively, an potential metastasis gene (Tiam-1) has been cloned byvirus-insertion mutagenesis (Habets G. G. M. et al., Cell 77:537-549,1994). However, many of genes isolated by such processes were notmetastasis-related genes, indicating that the efficiency of screeningfor metastasis-related genes is still low.

[0045] If a gene derived from a tumor cell could be introduced into1-1ras1000 cells of the present invention having tumorigenic potentialbut lacking invasive/metastatic potential to confer invasive/metastaticpotential on said cells, the introduced gene would greatly contribute tothe acquisition of invasive/metastatic potential. The above screeningmethod of the present invention can be used to efficiently identify agene conferring invasive/metastatic potential on tumor cells, andtherefore, it is very useful for investigating the mechanism ofmetastasis.

[0046] In the present invention, the v-src oncogene was actuallyintroduced into 1-1ras1000 cells to confirm that these cells haveacquired invasive/metastatic potential. Also v-src conferredinvasive/metastatic potential on the parent strain BALB/c 3T3 A31. Thesefacts suggest that the src gene is involved in conferringinvasive/metastatic potential.

[0047] Further in the present invention, DNA was extracted from tumorcell lines having various metastatic potentials and transfected intonon-metastatic 1-1ras1000 cells of the present invention, and thetransformed cells were injected into nude mice to evaluate metastaticpotential. As a result, the transformed cells were found to haveacquired metastatic potential, which was stable and unchanged even aftersubcultures. This demonstrated that 1-1ras1000 cells constitute avaluable screening tool for DNA having tumor metastatic potential.

[0048] Thus, the present invention also provides a set of a tumor cellline having tumorigenic potential but lacking invasive/metastaticpotential and a tumor cell line derived from the same parent strain andhaving both of tumorigenic potential and invasive/metastatic potential,which are very useful for investigating the difference between thesignaling pathway for acquiring tumorigenic potential and the signalingpathway for acquiring invasive/metastatic potential.

[0049] The screening method of the present invention can be used todiscriminate benign tumors from malignant tumors, predict theaggressiveness of metastasis or predict organ-specific metastasis.Moreover, genes identified as conferring invasive/metastatic potentialon tumor cells by the screening method of the present invention can beused to obtain metastasis-related proteins that are promising fordeveloping novel diagnostic agents or therapeutic agents for tumors. Thepresent invention has a very wide industrial applicability.

[0050] The following examples further explain the present invention indetail, but are not construed as limiting the scope thereof.

EXAMPLES

[0051] Strains and plasmids used in the examples of the presentinvention are at first briefly described below.

[0052] Strains

[0053] BALB/c 3T3 A31-derived clones 1-1 and 1-13 used as recipientcells in the present invention were established by Kakunaga (Kakunaga T.et al., Science 209:505-507, 1980), and are differentially susceptibleto radiations—and chemicals—induced neoplastic transformation. Here,early subcultures of the original cell stock were used. UV—and MCA(3-methylcholanthrene)—induced transformants (Tatsuka M., Nature359:333-336, 1992) and Ki-MSV (Kirsten murine sarcoma virus)-transformedcells (Kakunaga T., in Omen G. S. et al., (eds.), Genetic Variability inResponses to Chemical Exposure, Cold Spring Harbor Laboratory, New York,1984, pp. 257-274) were also used. All of those transformants weretested to be tumorigenic when subcutaneously injected into nude mice.The negative and positive control cells used in experiments forassessing invasive/metastatic potential were 3Y1 and fos-SR-3Y1-202,respectively (Taniguchi S. et al., Cancer Res. 49:6738-6744, 1989).

[0054] All the strains were cultured in EMEM supplemented with 10% FCSin 5% CO₂ at 37° C.

[0055] Plasmids

[0056] pSV2neo-ras contains the activated c-Ha-ras inserted into theBamHI site of pSV2neo. pcDsrc contains the v-src oncogene inserted intothe EcoRI site of pcDsrc (Yagi T. et al., Mol. Carcinog. 1:222-228,1989; Kizaka et al., Mol. Cell. Biol. 9:5669-5675, 1989). The plasmidpHyg carrying a hygromycin-resistant gene was also used.

[0057] Example 1: Transformation of BALB/c 3T3 A31 Variant Cells

[0058] BALB/c 3T3 A31 variant cells established by Kakunaga, clone 1-1(hereinafter sometimes referred to as A31-1-1) and clone 1-13(hereinafter sometimes referred to as A31-1-13), were examined for theirsusceptibility to in vitro transformation induced by UV-irradiation,MCA-exposure and Ki-MSV carrying the v-Ki-ras oncogene as describedabove.

[0059] Focus-Forming Ability of Transformants

[0060] A31-1-13 was more susceptible to the chemically and physicallyinduced neoplastic transformation than A31-1-1, as previously reported(Tatsuka M. et al., supra. 1992). Both clones showed similarsusceptibility to the Ki-MSV-induced transformation, as previouslyreported (Kakunaga T., supra. 1984). Morphology of the foci differedbetween both clones but not with the carcinogens for the inducedtransformation. The foci were larger and more aggressive in A31-1-13than in A31-1-1.

[0061] Tumorigenicity and Invasive or Metastatic Potential ofTransformants

[0062] All the transformants were tested to form tumors whensubcutaneously injected into nude mice or newborn BALB/c mice while notumor was formed with the parental cells and Mock transfected cells (forexample, cells transformed in the absence of only MCA). Growth rate andlatency period did not differ significantly between both clones.

[0063] Since activated ras oncogenes are known to often induceinvasive/metastatic potential in transformed cells, the transformantscarrying an activated ras oncogene were injected into the tail vein ofnude mice to examine whether they form metastatic lesions in lung.

[0064] Throughout the present invention, tumorigenicity and experimentalmetastasis assays were performed in the following manner.

[0065] BALB/c nude mice at 6-7 weeks of age were provided. Cells werecollected after trypsinization and washed with PBS. For tumorigenicityassay, 10⁶ cells were injected subcutaneously into the nude mice. Forexperimental metastasis assay, 5×10⁵ cells were injected into the tailvein of 7-8 week-old nude mice. The mice injected with ras-transformedcells were sacrificed after 5 weeks and autopsied for metastases.However, preliminary experiments had showed that mice injected withsrc-transformed cells die in 18-24 days as a result of aggressive growthof lung metastatic tumor cells. To avoid this phenomenon, the miceinjected with src-transformed cells were sacrificed and autopsied formetastases at the 2nd week. Metastasized lung nodules were counted afterinsufflation of lung with 15% Indian ink (Wexler H., J. Natl. CancerInst. 36:641-645, 1966).

[0066] In some experiments, BALB/c mice of wild type or with suppressednatural killer (NK) cells were used. To eliminate NK activity, 200 μl ofthe anti-asialo GM₁ serum was injected into the tail vein of 7-8week-old BALB/c mice for 3 days prior to injection of tumor cells. Theanti-asialo GM₁ serum is known to specifically bind to NK cells tosuppress their function (Kasai M. et al., Eur. J. Immunol. 10:175-180,1980).

[0067] The results of tumorigenicity and invasive/metastatic potentialobtained from A31-1-1 cells and A31-1-13 cells are shown in thefollowing Table 1. TABLE 1 Metastasis Assay in A31-1-1 and A31-1-13Cells Transformed with UV, MCA and Ki-MSV No. of No. of mice mice givenNo. of with nodues Cell line injections nodules (nodules/mouse) CellsTransformed by UV* 1-1UV 5† 0 0 1-13UV 5† 0 0 Cells Transformed by MCA*1-1MCA 5† 0 0 1-13MCA 5† 0 0 Cells Transformed by Ki-MSV* 1-1ki-msv1 5†0 0 1-1ki-msv2 5† 0 0 1-1ki-msv3 5† 0 0 1-1ki-msv4 5† 0 0 1-1ki-msv5 5†0 0 1-13ki-msv1 5† 0 0 1-13ki-msv2 5† 0 0 1-13ki-msv3 5† 0 0 1-13ki-msv45† 0 0 1-13ki-msv5 5† 0 0 Positive Control 3‡ 324 3 (149,103,72)fos-SR-3Y1-202 Negative Control 5† 0 0 3Y1 # nude mice.

[0068] † 5×10⁵ cells of each line were intravenously injected into thetail vein of BALB/c nude mice. After 5 weeks, the animals weresacrificed and autopsied for lung metastasis.

[0069] ‡ The animals were autopsied at 2 weeks.

[0070] As shown from Table 1, no lung invasive/metastatic potential wasobserved in transformed cells. When rat 3Y1 cells transformed with v-srcand v-fos (fos-SR-3Y1-202) were injected as a positive control, a numberof metastatic lesions were found in lung.

[0071] Thus, BALB/c 3T3 A31 variant cells, A31-1-1 and A31-1-13, werefound to be susceptible to neoplastic transformation by different kindsof carcinogens such as UV irradiation, chemicals and ras oncogenes.Although the transformants were tumorigenic, their invasive/metastaticpotentials examined by experimental invasion/metastasis assay werenegligible.

[0072] Example 2: Transfer of the Activated c-Ha-ras Oncogene intoBALB/c 3T3 A31 Variant Cells

[0073] To isolate cells expressing an activated Ras oncoprotein at ahigh level, A31-1-1 and A31-1-13 were transformed by introducing aplasmid containing the activated c-Ha-ras (derived from T24 bladdercarcinoma cells) and a selectable marker neo (pSV2neo-ras).Transformation was performed by electroporation (Tatsuka M., et al.,Exp. Cell Res. 178:154-162, 1988). The transformants were selected inthe presence of 400 and 1000 μg/ml of G418. Two clones, 1-1 ras and 1-13ras, were isolated in the presence of 400 μg/ml of G418 and other twoclones, 1-1ras1000 and 1-13ras1000, were isolated in the presence of1000 μg/ml of G418. All the isolated clones had similar properties tothose shown by cells transformed with v-Ki-ras. These cells were usedfor the following experiments.

[0074] Southern Blot Analysis of Incorporated Gene

[0075] Genomic DNA from each ras-transformed cell was digested with therestriction enzyme SacI and transferred to a nitrocellulose membrane byusing the BIO-DOT SF apparatus (Bio-Rad). The membrane was hybridizedwith the ³²P-labeled ras probe and detected on X-ray film. The copynumber of the introduced gene was determined by densitometry. As aresult, the ras gene introduced into cells was detected as a 3.0-kb SacIband.

[0076] Northern Blot Analysis of Transcribed mRNA

[0077] Cytoplasmic RNA was prepared by guanidiniumthiocyanate-chloroform extraction (Chomczynski P. et al., Anal. Biochem.162:648-657, 1977). Blotting and hybridization were performed asdescribed by Maniatis et al. (Maniatis T. et al., Molecular Cloning: ALaboratory Manual, Cold Spring Harbor Laboratory, New York, 1982). Theintroduced c-Ha-ras and v-src genes were assayed for mRNA using each³²P-labeled specific probe. As a result, mRNA of ras was detected.

[0078] This confirmed that the ras gene had been integrated into thegene of cells and transcribed into mRNA.

[0079] Evaluation of the Copy Number of ras by Slot Blot Analysis

[0080] Each 5, 10 or 15 μg of genomic DNA digested with SacI washybridized with the ³²P-labeled SacI/SacI fragment of the c-Ha-ras genederived from T24 human bladder carcinoma by slot blotting. The resultsare shown in FIG. 1. The copy number was higher in 1-1ras1000 and1-13ras1000 cells than in the cells selected in the presence of 400μg/ml of G418 (16- and 4-times, respectively).

[0081] Immunoblotting Analysis of p21^(ras)

[0082] The expression level of Ras oncoprotein was examined byimmunoblotting using an anti-p21^(ras) monoclonal antibody.

[0083] Pelleted cells were lysed in Laemmli's sample buffer (LaemmliU.K., Nature 227:680-685, 1979) by a brief sonication. The proteinconcentration was adjusted to 2 mg/ml. The lysates were heated at 100°C. for 3 min, and loaded onto a 15% polyacrylamide gel. Proteins in thegel were electroblotted onto Durapore filters (Towbin H. et al., Proc.Natl. Acad. Sci. USA 76:4350-4354, 1979) and incubated for 18 h with ananti-p21^(ras) monoclonal antibody, NCC-RAS-004 (Kanai T. et al., Jpn.Cancer Res. 78:1314-1318, 1987). This antibody can detect the p21^(ras)protein transcribed by the c-Ha-ras gene with a point mutation at thecodon 12. During then, specific binding on membranes was blocked withskimmed milk. The proteins transferred on the membranes were furtherincubated with ¹²⁵I-labeled Protein G (Akerstrom B. et al., J. Immunol.135:2589-2592, 1984) for 1.5 h, then autoradiographed on Fuji RX X-rayfilm.

[0084] The results are shown in FIG. 2. A rapidly migrating weak bandcorresponds to the endogenous p21^(ras). A slowly migrating band of thep21^(ras) oncoprotein translated from the mutated c-Ha-ras gene(Taparowski E. et al., Nature 300:762-765, 1982) was detectedspecifically in the ras-transformed cells. Such a slowly migrating bandof the p21^(ras) oncoprotein was never detected even when 10-fold excess(50 μl) of the parental cell lysate was electrophoresed on the gel (FIG.2).

[0085] Evaluation of Tumorigenicity and Invasive or Metastatic Potentialof the Cells Transformed with c-Ha-ras and v-src

[0086] To examine whether or not an increased expression level of thep21^(ras) oncoprotein affects the invasive/metastatic potential oftransformants, the cells transformed with c-Ha-ras and v-src were testedfor their tumorigenicity and invasive/metastatic potential. The resultsare shown in the following Table 2. TABLE 2 Tumorigenicity and Invasiveor metastatic Potential in A31-1-1 and A31-1-13 Cells Transformed withc-Ha-ras and v-src Oncogenes Tumorigenicity Metastatic potential No. ofNo. tumor bearing of mice No. of mice mice/No. of given No. of withnodules Cell line injected mice injections nodules (nodules/mouse)Parental Variant Cells or Cells Transfected with Empty Vector 1-1 0/5*5‡ 0 0 1-13 0/5 5‡  0 0 1-1neo ND 3‡  0 0 1-13neo ND 3‡  0 0 CellsTransfected with Mutated c-Ha-ras1 Oncogene 1-1ras4 3/3† 5‡  0 01-1ras1000 3/3† 5‡  0 0 1-13ras2 3/3† 5‡  0 0 1-13ras1000 3/3† 5‡  0 0Cells Transfected with v-src Oncogene 1-1src, 313† 3§ 154 3 (58,48,48)pooled cells 1-1src,clone1 ND 3§ 286 3 (126,89,71) 1-1src,clone2 ND 3§213 3 (94,67,52) 1-1src,clone3 ND 3§ 316 3 (118,116,82) 1-13src, 3/3† 3§172 3 (69,53,50) pooled cells 1-13src, ND 3§ 203 3 (85,77,41) clone11-13src, ND 3§ 342 3 clone2 (134,105,103) 1-13src, ND 3§ 265 3(104,92,69) clone3 Additional Transfection with v-src Oncogene or EmptyVector to 1-1ras1000 Cells 1-1ras1000hyg 3/3† 6‡ 0 0 1-1ras1000src 3/3†9§ 1227 9(196,165,146, 138,130,124 116,112,100) # after injection. #animals, tumor weight was 9-10 g after 3 weeks.

[0087] As shown from Table 2, the increased expression level of thep21^(ras) oncoprotein did not affect at all the invasive/metastaticpotential of transformants.

[0088] This revealed that the invasive/metastatic phenotype of theras-transformed cells was very stable and was hardly changed during invitro cultivation.

[0089] Example 3: Invasive or Metastatic Potential of BALB/c 3T3 VariantCells can be Induced by a Viral Oncogene v-src

[0090] A viral oncogene, v-src, has a strong transforming activity andcan induce invasive/metastatic potential simultaneously, depending onthe type of the recipient cell. Thus, the effect of v-Src oncoprotein onthe parental BALB/c 3T3 A31 variant cells as well as on theras-transformed cells was examined by the transfection with thisoncogene.

[0091] Upon transfection with v-src, the parental cells (BALB/c 3T3 A31cells) formed foci in vitro, indicating that the v-src gene itself hasthe activity of transforming cells. The shape of the focus-formingtransformed parental cells was not so different from that of the cellstransformed with the mutated ras gene.

[0092] Then, the transformants were confirmed to contain the transfectedv-src gene as a 2.9-kb EcoRI fragment by Southern blotting and afunctional expression of v-Src oncoprotein was also confirmed asfollows.

[0093] Immunoblotting Analysis and Tyrosine Kinase Assay of p60^(src)

[0094] The expression level of v-src gene products in src-transformantswere examined by immunoblotting analysis of p60^(src) usinganti-p60^(src) serum specific to Src protein (Cooper J. A. et al., J.Virol. 48:752-764, 1983). The src-transformants were also examined forphosphorylation of Src protein by the kination assay as described byJove et al. (Jove R. et al., J. Virol. 60:849-857, 1986).

[0095] As a result, it was difficult to distinguish v-Src from theendogenous c-Src by immunoblotting analysis (FIG. 3A). However, anenhanced autophosphorylation of the p60^(src) was observed in thetransformed cells by kination assay following immunoprecipitationexperiment (using anti-p60^(src) serum) (FIG. 3B).

[0096] src-Transformed Cells Have Acquired Tumorigenicity and Invasiveor Metastatic Potential

[0097] A pooled fraction of the src-transformed cells derived fromA31-1-1 and A31-1-13 efficiently formed tumors within 1-2 weeks afterinjection, and the latency period was the same as that of theras-transformed cells. The pooled transformants and each transformedclone (1-1src clones 1 to 3, 1-13src clones 1 to 3, and 1-1ras1000src)formed a number of lung metastases by the experimentalinvasion/metastasis assay (Table 2).

[0098] Thus, a clear difference was observed between the activated ras-and v-src-transformed cells in their invasive/metastatic potentialanalyzed by the experimental invasion/metastasis assay. In order toexamine whether or not this difference results from the sensitivity ofthe ras-transformed cells to nude mice, metastatic potentials of theras-transformed 1-1ras1000 cells and src-transformed 1-1 src cells werecompared using wild-type and NK-suppressed BALB/c mice instead of nudemice. This yielded the same results in either wild-type or NK-suppressedmice (FIG. 4), indicating that the above difference in metastaticpotential is not due to the sensitivity to NK activity in nude mice.

[0099] Example 4: In vitro Invasiveness and Cell Motility of the ras-and src-Transformed Cells

[0100] In vitro cell motility and invasiveness are known to be closelyrelated to the invasive/metastatic potential of tumor cells. An invasiveability of cells was analyzed by the method of Albini et al. (Albini A.et al., Cancer Res. 47:3239-3245, 1987) using a modified Boyden chamberfitted with a Matrigel-coated filter. As a result, v-src transformedcells A31-1-1 and A31-1-13 showed invasion indices 4-5 times higher thanthose of the ras-transformed cells (FIG. 5A). The invasiveness was notaffected by an increased expression level of the mutated Rasoncoprotein.

[0101] Cell motility was measured on Matrigel-coated dishes usingcomputer-aided digital image analysis (Tatsuka M. et al., Exp. Cell Res.185:342-352, 1989) and indicated by the motility index. As a result, themotility indices of the src-transformed cells were also 6-7 times higherthan those of the ras-transformed cells (FIG. 5B). This suggests thatv-Src oncoprotein induces various additional cellular phenotypes thatare not induced by the activated Ras protein, and consequently thesrc-transformants become invasive/metastatic.

[0102] Example 5: Induction of Metastatic Potential by DNA Derived fromHuman Tumor Cells

[0103] DNA extracted from various human metastatic tumor cells wastransfected into non-metastatic 1-1ras1000 cells to examine induction ofmetastatic potential.

[0104] A. Experimental Procedures

[0105] (1) Extraction of DNA

[0106] The following human tumor cell lines were used as experimentalmaterials to extract whole genomic DNA according to the method ofManiatis et al. (Maniatis, T. et al., Molecular Cloning: A LaboratoryManual, Cold Spring Harbor Laboratory, New York, 1982).

[0107] A204 (ATCC HTB-82): rhabdomyosarcoma;

[0108] Malme-3M (ATCC HTB-64): lung-metastasized malignant melanoma;

[0109] Mewo (JCRB0066): malignant melanoma;

[0110] SW480 (ATCC CCL-228): colon adenocarcinoma.

[0111] These cells were obtained from American Type Culture Collection(ATCC) or Japanese Collection of Research Biosources (JCRB).

[0112] The size of each DNA extracted from these cell lines exceeded20-90 kb, as determined by pulsed field electrophoresis.

[0113] (2) DNA Transfection

[0114] DNA transfection was performed by the calcium phosphate method(Graham, F. L. and Van Der EB, A. J., Transformation of rat cells by DNAof human adenoma 5, Virology, 96:64-79, 1973). Prior to transfection,5×10⁵ 1-1ras1000 cells were seeded in dishes of 100 mm in diameter. Eachgenomic DNA sample prepared by mixing 1 mg of genomic DNA with 100 μg ofpSV2gptDNA (dominant selectable marker) was precipitated and added in 20dishes. After 4 hours, cells were shocked with 15% glycerol for 60 sec.Cells were grown for 16 h in Eagle's MEM supplemented with 10% fetalbovine serum. Then, a selective medium containing xanthine (250 μg/ml),hypoxanthine (15 μg/ml), thymidine (10 μg/ml), aminopterin (2 μg/ml) andmycophenolic acid (MPA: 25 μg/ml) was added to each dish. MPA-resistantcolonies which appeared on dishes were pooled for further experiments.

[0115] (3) Evaluation of Metastatic Potential

[0116] Male BALB/c nude mice were obtained at 6-7 weeks of age.MPA-resistant cells were collected by trypisinization. The pooled cellsfrom each transfection were washed with Hanks' solution and 5×10⁵ cellswere injected into the tail vein of 7-8 week-old nude mice. The animalswere sacrificed after 5 weeks and autopsied. Cells were obtained byculture of minced lung. If a metastatic lesion was formed in lung, acolony appeared upon culture on an MPA-selective medium. Normally, acolony was generated from each focus metastasized to the lung of onemouse. The number of colonies was counted and all the colonies from eachmouse were used for a second round of transfection.

[0117] B. Results

[0118] MPA-resistant colonies incorporating DNA from the cell line A204,Malme-3M, Mewo or Sw480 metastasized to lung (Table 3). MPA-resistantcolonies recovered from the lung with metastases were pooled andcultured, and then reinjected into mice. As a result, all the coloniesmetastasized to lung (Table 3).

[0119] A pooled strain of 1-1ras1000 cells incorporating DNA of SW480was used for further transfection experiments. When DNA extracted fromthis strain was retransfected into 1-1ras1000 (second transfection),those cells metastasized to lung. When the same procedure was repeatedagain (third transfection), lung metastasis was observed. The number ofcolonies metastasized to lung increased with an increase of rounds oftransfection (Table 3). Transformed cells were stable with no change inmetastatic potential after at least 5 passages in normal medium. TABLE 3No. of mice given No. of colonies DNA tranfected i.v. administrationobserved per mouse First transfection 1-1ras1000 5 0,0,0,0,0 A204 53,4,12,15,18¹ Malme-3M 5 0,5,6,8,9² Mewo 5 0,0,0,1,2³ SW480 5 0,3,5,6,8⁴Second transfection 1-1ras1000 6 0,0,0,0,0,1 1-1ras1000 SW480-T1 615,20,24,34,48,63⁵ Third transfection 1-1ras1000 4 0,0,0,0 1-1ras1000SW480-T2 4 23, >200, >200, >200 # nude mice. # nude mice. # nude mice.

1. A tumor cell having tumorigenic potential but lackinginvasive/metastatic potential.
 2. The tumor cell according to claim 1 ,wherein the tumorigenic potential has been conferred by an oncogene ofthe ras family.
 3. The tumor cell according to claim 2 , wherein theoncogene of the ras family is the c-Ha-ras gene.
 4. The tumor cellaccording to any one of claims 1 to 3 , which is 1-1ras1000 (accessionnumber FERM BP-5406).
 5. A method for preparing the tumor cell accordingto claim 1 by introducing an oncogene of the ras family into a BALB/c3T3 A31 variant cell.
 6. The method according to claim 5 , wherein theBALB/c 3T3 A31 variant cell is BALB/c 3T3 A31-1-1 or BALB/c 3T3A31-1-13.
 7. The method according to claim 5 , wherein the oncogene ofthe ras family is the c-Ha-ras gene.
 8. The method according to any oneof claims 5 to 7 , wherein the tumor cell is 1-1ras1000 (Accession No.FERM BP-5406).
 9. A screening method for genes having the property ofconferring invasive/metastatic potential, which comprises transfectingDNA derived from a tumor tissue obtained from the surface or inside of amammal or derived from a tumor cell line into the tumor cell accordingto claim 1 , isolating cells having acquired invasive/metastaticpotential and extracting DNA therefrom.
 10. The method according toclaim 9 , wherein DNA is a genomic gene of a metastatic tumor cell or acDNA library prepared with a mammalian cell expression vector.
 11. Themethod according to claim 9 or 10 , wherein the tumor cell transfectedwith DNA is 1-1ras1000 (Accession No. FERM BP-5406).